Adeno-associated viruses (AAVs) are a group of naturally defective parvoviruses that are being developed as gene therapy vectors for the treatment of diabetes, obesity, diabetes-associated erectile dysfunction, cystic fibrosis, hemophilia B, and glycogen storage diseases. AAVs require co-infection with a helper virus, usually an adenovirus or herpesvirus, for efficient productive infection. In the absence of helper virus, AAV type 2 (AAV2) DNA can integrate into the host genome with a strong preference (70%-90% of integration events) for a 4 kb region of human chromosome 19, designated AAVS1 (the only example of site-specific integration in a mammalian virus system). This ability to preferentially integrate also contributes to AAV2's attractiveness as a vector for gene therapy, since this could potentially limit the dangers associated with insertional mutagenesis. The rep gene of AAV2 encodes Rep proteins that are required for preferential integration, replication and packaging. First generation AAV-based gene therapy vectors have deleted the rep gene, both to make more space for therapeutic genes and to limit the generation of wild-type virus, through recombination, during virus production. To allow the use of the rep gene, with its potential for directing preferential integration, we are developing a dual vector transduction system, in which cells would be co-infected with one vector containing the rep gene and a second vector containing the therapeutic gene. As an obligatory step in the creation of this system, we have developed a packaging system for rep-positive AAV vectors that should prevent the production of wild-type AAV. This was an especially difficult challenge since there are approximately 400 bases of overlap between the rep gene and the gene encoding the virus capsid. Gene therapy for chronic diseases, such as diabetes, requires long-term expression of therapeutic genes. Although AAV vectors without a rep gene are capable of expressing genes from non-dividing cells for greater than a year, data suggests that these rep-minus AAV vectors have a lower integration frequency than wild-type AAV and episomal rep-minus AAV genomes appear to be lost rapidly in dividing cells. Although the Rep proteins have the potential to increase the integration frequency (compared to vectors without a rep gene), when expressed at high levels, they can inhibit cell division and may even induce apoptosis, which could lead to a net reduction in the number of actively dividing cells expressing a therapeutic gene. Dividing cells, such as those that replenish the beta cells of pancreatic islets, are prime targets for diabetes gene therapy. We have therefore transfected plasmids containing recombinant AAV2 genomes with a neomycin/G418 resistance gene (driven by the AAV2 p40 [capsid gene] promoter), plus either a wild-type or defective AAV2 rep gene (driven by its natural promoter, p5) into various human cell lines. With the four cell lines tested thus far, we saw no reproducible difference in the size or number of G418-resistant colonies following transfection with the rep-plus versus the rep-minus plasmid. We interpret these results as indicating a general lack of rep gene-induced toxicity, when the gene is controlled by its natural promoter.